Cyclic AMP-dependent protein kinase (PKA) is anchored at specific subcellular sites via the interaction of the regulatory subunit (R) with A-kinase anchoring proteins (AKAPs) via an amphipathic helix binding motif. Synthetic peptides containing this amphipathic helix domain competitively disrupt PKA binding to AKAPs and cause loss of PKA modulation of cellular responses. We have recently shown that S-Ht31, a cell permeant anchoring inhibitor peptide (AIP), is able to arrest mammalian sperm motility. In this report we identify two tyrosine phosphorylated proteins whose phosphorylation levels are regulated by S-Ht31. The phosphorylation levels of one of these proteins, a 55 kDa protein found in the soluble fractions, correlated with the level of sperm motility. Immotile caput sperm have low levels of tyrosine phosphorylation of this protein compared to vigorously motile caudal sperm. Agents which increase motility, such as Isobutyl Methyl Xanthine IBMX and 8-Bromo-cAMP (8-Br-cAMP), increase tyrosine phosphorylation of the 55 kDa protein while agents which decrease motility, such as S-Ht31 and ionomycin plus calcium, completely abolish tyrosine phosphorylatin of the 55 kDa protein. This is the first documentation of a protein whose tyrosine phosphorylation parallels motility. Treatment of sperm with S-Ht31 also affects the tyrosine phosphorylation of a 39 kDa protein found in the insoluble fraction. In this case, S-Ht31 causes a dramatic increase in phosphorylation, suggesting that sperm PKA anchoring affects a substrate specific tyrosine kinase and/or phosphatase which may be involved in the regulation of sperm motility.